Lactate is a key intermediate during anaerobic digestion of carbohydrates; however, it fails to receive significant consideration in biogas plants. We examined the influence of lactic acid on biogas production. Two commonly used feeds, fresh maize and maize silage, were selected as substrates due to their difference in lactic acid contents. Additionally, inocula from an agriculture-based biogas plant, a waste water treatment plant and a standardised laboratory reactor were selected to investigate the impact of starter culture on the process. Experiments demonstrated increased total biogas yield of up to 45% in the lactate-rich maize silage over the lactate-devoid fresh maize, but only in cases where the starting inocula had been previously exposed to lactic acid. Our findings suggest lactic acid is a significant intermediate in biogas production and merits consideration. Additionally, the ability of the starter inoculum to utilize lactic acid is an important factor in process optimization and enhanced biogas production.
Industry creates large quantities of nutrient-rich effluents that, if released into the environment, can cause environmental pollution and disturb the ecosystem [
Lactic acid has been described previously in literature as a significant acid generated in the anaerobic digestion of ensiled energy crops such as maize silage or fermented vegetable wastes [
Biogas reactor failures have been reported previously in acidification reactors during transient overload conditions as a direct result of lactic acid accumulation [
Maize and its silage are the most ensiled crops in the world, widely used for biogas generation due to their high carbohydrate content and easy cultivability [
Anaerobic bacteria have a major role in the fermentation process, particularly in the hydrolysis of carbohydrates to monosaccharides. These monosaccharides are degraded to lactic acid, which is then converted to H2 and acetate for further conversion to biogas [
The structure and activity of the microbial community involved in fermentation often depends on the original inoculum source and starter biomass, as well as the operational and environmental conditions [
Three different inocula were selected for testing in this study; one from an agriculture-based biogas plant, one from a waste water treatment plant, and a final inoculum from a continually controlled and monitored standard reactor. The aim of this research was to investigate the role of starter inocula, lactic acid and substrate type on biogas yield, with a view to improving processes and predictive models of biogas systems.
Fresh maize and maize silage were sourced from the same farm in the Emden region of Lower Saxony, Germany. Fresh maize plant was freshly harvested and roughly shredded into 1 cm pieces and prepared maize silage was collected from the same farmer. All samples were frozen at −20˚C for later analysis.
The agriculture biogas plant bacterial inoculum was obtained from EWE biogas plant, Wittmund, Lower Saxony, Germany [
The wastewater treatment plant inoculum was obtained from the anaerobic digester of the municipal sewage treatment plant of Papenburg, Lower Saxony, Germany [
A standard inoculum was prepared in a 12 L Plexiglass Continuous Stirred Tank Reactor (CSTR) with a working volume of 8 L, and was maintained at 38˚C. Initial inoculum was sourced from the EWE Wittmund biogas plant [
Feed type | Component | Quantity |
---|---|---|
Energy sources | Starch | 20 g |
Rapeseed oil | 30 g | |
Casein protein | 30 g | |
A) Nutrient solution | NH4Cl | 100 g/L |
NaCl | 10 g/L | |
MgCl2∙6H2O | 10 g/L | |
CaCl2∙2H2O | 5 g/L | |
B) Trace-metal and selenite solution | FeCl2∙4H2O | 2 g/L |
H3BO3 | 0.05 g/L | |
ZnCl2 | 0.05 g/L | |
CuCl2∙2H2O | 0.038 g/L | |
MnCl2∙4H2O | 0.05 g/L | |
(NH4)6Mo7O24∙4H2O | 0.05 g/L | |
AlCl3 | 0.05 g/L | |
CoCl2∙6H2O | 0.05 g/L | |
NiCl2∙6H2O | 0.092 g/L | |
WOCl4 | 0.04 g/L | |
Ethylenediaminetetraacetate | 0.5 g/L | |
Concentrated HCl | 1 mL/L | |
Na2SeO3∙5H2O | 0.16 g/L | |
C) Vitamin mixture | Biotin | 2 mg/L |
Folic acid | 2 mg/L | |
Pyridoxine acid | 10 mg/L | |
Ridoflavin | 5 mg/L | |
Thiamine hydrochloride | 5 mg/L | |
Cyanocobalamine | 0.1 mg/L | |
Nicotinic acid | 5 mg/L | |
P-aminobenzoic acid | 5 mg/L | |
Lipoic acid | 5 mg/L | |
DL-pantothenic acid | 5 mg/L | |
Sørenson’s buffer | Na2HPO4 | 0.133 M |
KH2PO4 | 0.133 M |
was adjusted to a final pH of 7.4 with NaOH. The prepared medium was further required to be free from contamination during feeding and storage which was achieved by reducing the pH to 4.0 using 5 - 6 mL formic acid (conc. ≥ 95%). This prepared standard medium was fed into the reactor hourly, with a retention time of 18 days.
The inoculum collected from this reactor was used for experimentation after 5 volume changes to ensure no influence of the EWE biogas plant starter microbial cultures. The 16S rRNA sequencing was undertaken to ensure a difference in the microbial community composition of the starter inoculum from EWE biogas plant the final standard reactor population. The standard bacterial inoculum was grown in this continuous culture and was collected from the outlet of the reactor. RITTER Milligas® counter provided continuous monitoring of biogas production, and methane and carbon dioxide was measured via BlueSens gas sensors from the outlet port of the reactor.
Known quantities of sample were taken and dried in an oven at 105˚C. The volatile solids were quantified by combusting dried samples at 550˚C for 4 h [
D-and L-lactic acid were determined using enzymatic test kits from R-Bio- pharm. Sample preparation included filtering for inocula samples, and Carrez clarification for maize samples. Tests were then performed as per manufacturer’s instructions. The limits of detection by the enzymatic test were 1 - 60 mg/L.
A series of volatile fatty acids (VFA) standards for the calibration curves were prepared for Gas Chromatographic (GC) analysis using seven aqueous solutions of acids: acetic, propionic, butyric, isobutyric acid, valeric acid, isovaleric acid and lactic acid. These calibration samples were centrifuged for 3 minutes and adjusted to pH 2 using 0.1 N HCl, and spanned a concentration range of 1 to 100 g/L. GC analyses were performed on a GC 7820 Agilent gas chromatograph equipped with a flame ionization detector and an INNOPEG-FFAP column (30 m, 0.25 mm I.D., 0.25 μm film thicknesses, CS, Germany). This column is a high polar polyethylene glycol column suitable for separation of free fatty acids in aqueous extracts. The analyses were performed using an isothermal temperature program with the injector and detector temperatures maintained at 180˚C. The carrier gas was nitrogen. In each case a 1 μL of sample was injected with a flow splitting 1:50. Samples for analysis were centrifuged for 3 minutes and adjusted to pH 2 using 0.1 N HCl.
Batch experiments were prepared in accordance with the German standard VDI 4630 [
The three inocula were checked for the biogas production with lactic acid as the sole substrate. D/L-lactic acid from Sigma Aldrich was used for the experiments to check for its degradation with the different sources of inoculum. Batch experiments were performed with 10 mM (0.9 g/L) of lactic acid using ANKOM wireless gas monitors as described previously. The average values for biogas production were presented. Lactic acid with its strong pKa value showed a remarkable impact by decreasing the pH of the inoculum. Therefore, the pH was adjusted to the original pH for each inoculum using NaOH. Gas chromatographic analyses were performed daily with samples taken from the side vent of the bottle.
Primary reactor feed used for the standard reactor was optimized to attain maximal biogas production in batch experiments. The energy source concentration required to achieve maximal biogas production was maize starch, rapeseed oil and casein protein with the concentrations 1.3 g/L, 2.0 g/L and 2.0 g/L respectively. Any concentration of energy sources above these proved to be inhibitory to biogas production. A lactic acid concentration of 10 mM neutralized D/L-lactic acid was used to demonstrate the impact of lactic acid as a sole substrate. These were made up to a final volume of 100 mL with standard inoculum for the comparative study. Experiments with 150 mM and 250 mM lactic acid were further performed in order to determine the inhibitory concentration. The reference for this experiment was 100 mL of standard inoculum. Batch experiments were performed for 3 hours using ANKOM wireless gas monitors. An activity curve was created in order to infer an approximate contribution of a small fraction of lactic acid to the total activity of the standard reactor with the optimized feed. The experiments were performed in triplicates, with readings determined every 5 minutes and the average presented.
Fresh maize and silage were considered for the experiments due to the major differences in their lactate content [
DM (%) | VS (% DM) | D-Lactic acid (% DM) | L-Lactic acid (% DM) | Acetic acid (% DM) | |
---|---|---|---|---|---|
Fresh maize | 26 | 95 | 0.005 | 0.004 | 0 |
Maize silage | 28.5 | 97.6 | 2.8 | 2.0 | 0.6 |
with this, minor amounts of acetic acid (accounting to nearly 12.5% of that of lactic acid) were detected in maize silage as well.
Inocula from agriculture-based biogas plant and the standard reactor showed the presence of L-lactic acid, suggesting these inocula may contain microbial populations adapted to survive in and utilise the substrate. Inoculum from the waste water treatment plant showed a negligible content of D- and L-lactic acid (
A volume of 3.1L biogas was generated per day, with a total methane content of 68%. In the steady state, the metabolite showing the highest concentration was acetic acid (20 mM) which is a direct precursor of methane. Propionic acid (3 mM) and lactic acid (1 mM) were also detected. Starch can be enzymatically hydrolyzed to glucose and further fermented to lactic acid by lactic acid bacteria [
The 16S rRNA gene fragments of both bacteria and archaea revealed a clear difference in the microbial community composition of the starter inoculum from EWE biogas plant had BacteroidesandMethanosarcina, and the final standard reactor population, which was comprised of Firmicutesand Methanomethylovorans as predominant groups [
In experiments using agriculture-based inoculum, maize silage showed enhanced biogas production compared with fresh maize (
Inoculum from | DM (%) | VS (% DM) | D-Lactic acid (% DM) | L-Lactic acid (% DM) | Acetic acid (% DM) |
---|---|---|---|---|---|
Agriculture-based biogas plant | 4.9 | 66.2 | 0.01 | 0.28 | 0.02 |
Wastewater treatment plant | 3.0 | 99.3 | 0 | 0.03 | 0.08 |
Standard reactor | 1.8 | 26.7 | 0.02 | 0.66 | 0.01 |
marily fermented to lactic acid which are further transformed to acetate and hydrogen that can be used for methane formation [
The inoculum from the waste water treatment plant failed to show any considerable difference in biogas production between the substrates (
More biogas was produced from silage than fresh maize in the standard reactor experiments (
The reference samples had also shown substantial biogas production in the absence of added fresh maize and maize silage. This could be due to the intermediate organic acid contents in those samples. Acetic, propionic, lactic, butyric and valeric acids were detected in all inocula (presented in Appendix II). Acetic acid was found to be dominant in each case, with concentrations in the range of 0.5 - 1.6 g/L across each of the inocula. The biogas produced during batch experiments with these reference samples was subtracted and is presented (
Inoculum source | Biogas production from fresh maize (mL) | Biogas production from maize silage (mL) | Difference in biogas production with silage and fresh maize (mL) |
---|---|---|---|
Agriculture-based biogas plant | 1010 | 1838 | 838 |
Waste water treatment plant | 107 | 101 | −6 |
Standard Reactor | 85 | 142 | 57 |
Factors such as pH, alkalinity, volatile fatty acids (VFA) have also been identified to influence the biogas production [
Maize and silage differ significantly in terms of lactic acid content as discussed previously. The silage is preserved against degradation by bacterial contamination as a result of the high concentration of lactic acid and the corresponding low pH, and also bears a more hydrolyzed structure [
Biogas production from lactic acid as a sole substrate was measured to assess the impact of the acid alone on the system. Biogas was produced from lactic acid in cases where the microbial community present has the capacity to utilize the substrate. Maximum biogas production was observed by the inoculum from agriculture-based biogas plant (605 mL), followed by the inoculum from the standard reactor (460 mL) and the minimum biogas production observed in the inoculum from waste water treatment plant (210 mL) (
The inoculum from the waste water treatment plant displayed an inhibition of biogas production with no biogas production observed after an experimental run of 3 days. A reduced rate of lactic acid degradation was observed after 2 days, and GC analysis showed an accumulation of acetic and propionic acid. Propionic acid has been known to inhibit biogas production [
Lactic acid was not a component of standard reactor feed; however the bacteria present in the standard reactor were able to convert starch present in the feed to lactic acid which was further utilized for biogas production. As lactic acid is an intermediate of sugar degradation [
Biogas production from the optimized feed for standard reactor was determined to compare it with that from a small quantity of lactic acid (10 mM) and further with larger quantities (150 mM, 250 mM). Optimized reactor feed showing maxi-
mal biogas production was found to be 3 times the concentration fed into the standard reactor. This energy source combination was comprised of starch (1.3 g/L) mixed with rapeseed oil (2.0 g/L) and casein protein (2.0 g/L). All other components of standard reactor feed remained unaltered.
Maximum biogas production was observed in the first hour for all treatments. Optimized feed produced nearly 48% more biogas than the reference treatment. It was found that the addition of just 10 mM of lactic acid had a large impact on overall yield and production rate, producing almost 35% of the biogas of the optimized feed. A concentration of 150 mM lactic acid resulted in the largest increase in total biogas yield. This exceeded the overall biogas production capacity of the reactor with optimized feed. Inhibition of biogas production was observed at 250 mM (
Obviously, the bacteria in this standard reactor are able to utilize lactic acid, even at high concentrations (150 mM). This is then rapidly converted rapidly to biogas. The considerable impact of lactic acid on biogas production and the overall system has also been demonstrated by the research of others, particularly in relation to silage-based anaerobic digesters [
Lactic acid is an intermediate of carbohydrate degradation and its presence can have a significant impact on biogas production. As a result of the ensilaging process, maize silage often contains substantial quantities of lactic acid in comparison to fresh maize. Additionally, the partial fermentation of silage and the hydrolyzed structure can result in an advantage over fresh maize for increased biogas yields. Experiments demonstrated a 45% increase in total biogas yield
using maize silage over fresh maize, and lactic acid as a sole substrate can also result in an increased yield. This suggests that lactic acid plays an essential role in the increased total biogas production. These significant increases, however, were only observed in starter inocula which were previously exposed to lactic acid and that contained microbial communities able to utilize the intermediate. This finding is particularly important for facilities where lactic acid can be present in high quantities, such as silage, sugar and dairy-based biogas plants. Biogas reactor breakdowns have been reported in the past as a result of accumulation of lactic acid in their processes, therefore particular care must be taken when selecting a starter inoculum to ensure the utilization of this acid is possible. This research study could confirm that lactic acid is a key intermediate in biogas production, and provides rationale for its inclusion in predictive modeling and within the overall process. Our further research will focus on the impact of including lactate as a parameter in the Anaerobic Digestion Model No. 1 (ADM1) in influencing the prediction capability of the model.
This research was funded by Deutscher Akademischer Austauschdienst (DAAD. We thank Ingo Stein from EUTEC Institute, Hochschule Emden for his kind assistance. Engaged help of Candice Raeburn in proof-reading and improving the manuscript is gratefully acknowledged.
Satpathy, P., Steinigeweg, S., Siefert, E. and Cypionka, H. (2017) Effect of Lactate and Starter Inoculum on Biogas Production from Fresh Maize and Maize Silage. Advances in Microbiology, 7, 358-376. https://doi.org/10.4236/aim.2017.75030
Acetic acid content when mixed with the two substrates: maize, silage and without substrates(reference) in (a) Agricultural waste based reactor (b) Waste water treatment plant and (c) Standard reactor.
Organic acid content in the pure inoculum from (a) Agriculture-based biogas reactor (b) Waste water treatment plant and (c) Standard reactor.
Degradation of 10 mM lactic acid used as a solo substrate with the different inocula during batch experiments.
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